The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues, in vivo. Towards this goal, the laboratory concentrates on the use of minimally-invasive techniques to evaluate cellular energy metabolism of heart and skeletal muscle. The following major findings were made over the last year: 1) Blue native gel electrophoresis permits the separation of native protein complexes from cells and organelles. The intense color of the blue dye used in these studies interferes with analysis after the separation. We have developed a colorless analog of these dyes that retains the ability to separate the complexes with no color interference permitting many in gel assays. 2) Using native gel approaches we have found that other proteins associated with Complex 1, such as various dehydrogenases etc, will enhance NADH fluorescence within the native gels when studied under anaerobic conditions. These sites NADH binding sites associated with Complex 1 may represent the important entry steps of NADH into the initial steps of oxidative phosphorylation. The role of these Complex 1 associated dehydrogenases in oxidative phosphorylation is currently being evaluated. 3)A 32P screen of the mitochondrial matrix phospho-protein turnover has been expanded to evaluate the role of different experimental conditions on the rate of 32P incorporation into the matrix proteins. In these studies we have demonstrated the time course of 32P incorporation into all of the complexes involved in oxidative phosphorylation using the native gel electrophoresis techniques as well as the effects of anoxia and carbon substrate restriction. We have a focused effort on the F1-ATPase, the actual engine that generates ATP, were we have demonstrated the specific and dynamic phosphorylation of the alpha, beta, delta and gamma subunits. The functional consequences of these protein phosphorylations are currently being evaluated. These studies suggest that protein phosphorylation may play a much wider role in the regulation of mitochondrial matrix protein activity than previously appreciated. 5) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have continued to make improvements in the technology of this approach by modifying the telescoping system to permit rapid (KHz) focusing at the objective permitting the accurate collection of image plane stacks, as well as potentially track image planes within the animal. The scope has also been adapted to optimize the collection of light by placing the optical detector directly above the objective. This system is now being applied to an in vivo mouse skeletal muscle preparation where the work of the muscle can be quantitatively varied while directly observing the metabolic events occurring within the cells of the muscle. 6) We have also developed a new microscope to optimize the collection of signal from multiphoton excitation microscopy experiments. This new microscope collects all of the emitted light from the excited voxel in the multiphoton imaging experiment improving the signal to noise of these experiments by as much as a factor of 5. This improvement in signal to noise will improve the sensitivity of in vivo microscopy resulting in deeper detection of signal as well as increasing the time resolution of the imaging experiment by as much as 25 fold. This technology should contribute signficantly in the development of this technology to study intracellular events using intravital microscopy.